Apoptosis or programmed cell death is a genetically and biochemically regulated mechanism that plays an important role in development and homeostasis in invertebrates as well as vertebrates. Aberrancies in apoptosis that lead to premature cell death have been linked to a variety of developmental disorders. Deficiencies in apoptosis that result in the lack of cell death have been linked to cancer and chronic viral infections (Thompson et al., (1995) Science 267, 1456–1462).
Some of the key effector molecules in apoptosis are the caspases (cysteine containing aspartate specific proteases). Caspases are strong proteases, cleaving after aspartic acid residues and once activated, digest vital cell proteins from within the cell. Since caspases are such highly active proteases, tight control of this family of proteins is necessary to prevent premature cell death. In general, caspases are synthesized as largely inactive zymogens that require proteolytic processing in order to be active. This proteolytic processing is only one of the ways in which caspases are regulated. The second mechanism of regulation is through a family of proteins that bind and inhibit caspases.
A family of molecules that inhibit caspases are the Inhibitors of Apoptosis (IAP) (Deveraux et al., J Clin Immunol (1999), 19:388–398). IAPs were originally discovered in baculovirus by their functional ability to substitute for P35 protein, an anti-apoptotic gene (Crook et al. (1993) J Virology 67, 2168–2174). IAPs have been described in organisms ranging from Drosophila to human. Regardless of their origin, structurally, IAPs comprise one to three Baculovirus IAP repeat (BIR) domains, and most of them also possess a carboxyl-terminal RING finger motif. The BIR domain itself is a zinc binding domain of about 80 residues comprising 5 alpha-helices and 3 beta strands, with cysteine and histidine residues that coordinate the zinc ion (Hinds et al., (1999) Nat. Struct. Biol. 6, 648–651.; Sun et al., (1999) Nature 401, 818–22.; Sun et al., (2000) J. Biol Chem. 275,33777–81).
All IAP proteins contain one to three copies of the baculoviral IAP repeat (BIR), a zinc-binding domain of ˜80 amino acids, that are necessary for their interactions with a number of cytosolic target proteins, including activated caspases-3, -7, and -9, and natural IAP protein antagonists such as mature Smac/DIABLO and HtrA2/Omi. Different BIR domains, however, have differing affinities for these proteins, and thus distinct functions in the regulation of apoptosis. For instance, the second BIR domain of X-chromosome-linked IAP (XIAP) together with the immediately preceding linker region (XIAP-BIR2) binds to and inhibits caspases-3 and -7 with inhibition constants in the range of 2–10 nM (Takahashi et al., (1998) J Biol Chem 273(14): 7787–90.; Sun et al., (1999) Nature 401 (6755): 818–22), while the third BIR domain of XIAP (XIAP-BIR3) specifically inhibits caspase-9 with an inhibition constant in the range of 10–20 nM (Deveraux et al., (1999) Genes Dev 13: 239–252; Liu et al., (2000) Nature 408(6815): 1004–8.; Sun et al., (2000) J Biol Chem 275(43): 33777–81). By contrast, the single BIR domain of melanoma inhibitor of apoptosis (ML-IAP) has been shown to inhibit weakly caspases-3 and -9, but not caspase-7, although inhibition constants have not been reported (Vucic et al., (2000) Curr Biol 10(21): 1359–66).
Melanoma IAP (ML-IAP) is an IAP whose expression is strongly upregulated in melanoma (Vucic et al., (2000) Current Bio 10:1359–1366). Determination of protein structure demonstrated significant homology of the ML-IAP BIR and RING finger domains to corresponding domains present in human XIAP, C-IAP1 and C-IAP2. The BIR domain of ML-IAP appears to have the most similarities to the BIR2 and BIR3 of XIAP, C-IAP1 and C-IAP2, and appears to be responsible for the inhibition of apoptosis, as determined by deletional analysis. Furthermore, Vucic et al., demonstrated that ML-IAP could inhibit chemotherapeutic agent induced apoptosis. Agents such as Adriamycin and 4-tertiary butylphenol (4-TBP) were tested in a cell culture system of melanomas overexpressing ML-IAP and the chemotherapeutic agents were significantly less effective in killing the cells when compared to a normal melanocyte control. The mechanism by which ML-IAP produces an anti-apoptotic activity is through inhibition of caspase 3 and 9. ML-IAP did not effectively inhibit caspases 1, 2, 6, 7 or 8.
Since apoptosis is a strictly controlled pathway with multiple interacting factors, the discovery that IAPs themselves are regulated was not unusual. In the fruit fly Drosophila, the Reaper (rpr), Head Involution Defective (hid) and GRIM proteins physically interact with and inhibit the anti-apoptotic activity of the Drosophila family of IAPs. In the mammal, the proteins Smac/DIABLO act to block the IAPs and allow apoptosis to proceed. It was shown that during normal apoptosis, Smac is processed into an active form and is released from the mitochondria into the cytoplasm where it physically binds to IAPs and prevents the IAP from binding to a caspase. This inhibition of the IAP allows the caspase to remain active and thus proceed with apoptosis.
The proapoptotic function of these IAP protein antagonists is dependent on a conserved four-residue IAP protein-interaction motif (A-V/I-P/A-I/F/Y) found at the N-termini of the mature proteins Chai et al., (2000) Nature 406:855–862; Srinivasula et al., (2001) Nature 410(6824): 112–6). This conserved motif is also found at the N-termini of the Drosophila proteins Reaper, Hid, Grim, Sickle, and Jafrac2, that also antagonize IAP proteins and are thus functional homologues of Smac/DIABLO (White et al., (1994) Science 264(5159): 677–83.; Grether et al., (1995) Genes Dev 9(14): 1694–708; Chen et al., (1996) Genes Dev 10(14): 1773–82; Goyal et al., (2000) Embo J 19(4): 589–97; Christich et al., (2002) Curr Biol 12(2): 137–40; Srinivasula et al., (2002) Curr Biol 12(2): 125–30; Tenev et al., (2002) Embo J 21(19): 5118–29.; Wing et al., (2002) Curr Biol 12(2): 131–5.). Structural studies have shown that these N-terminal peptides bind to a surface groove on the BIR domains, with the binding being stabilized by electrostatic interactions involving the conserved N-terminal alanine residue of the peptide, together with several intermolecular hydrogen bonds and hydrophobic interactions (Liu et al., (2000) Nature 408:1004–1008, Wu et al., (2000) Nature 408 1008–1012; Wu et al., (2001) Mol. Cell 8, 95–104; Srinivasula et al., (2002) Curr. Biol. 12, 125–30; Franklin et al., (2003) Biochem. 42 8223–31).
Despite the above identified advances in apoptosis research, there is a great need for additional diagnostic and therapeutic agents capable of enhancing apoptosis in a mammal with the goal of inhibiting the progression of cancer. The present invention relates to a chimeric ML-IAP polypeptide in which certain residues correspond to those found in XIAP-BIR3, while the remainder corresponds to ML-IAP. The chimeric protein is shown to bind and inhibit caspase-9 significantly better than either of the native ML-IAP or XIAP, but binds Smac-based peptides and mature Smac with affinities similar to those of native ML-IAP. The improved caspase-9 inhibition of the chimeric ML-IAP polypeptide is correlated with increased inhibition of doxorubicin-induced apoptosis when transfected into MCF7 cells. Accordingly, the present invention relates to use of the ML-IAP chimeric polypeptide and methods for screening for IAP antagonists.